Kang MOLECULAR PAIN RESEARCH Open Access N-type voltage ... · RESEARCH Open Access N-type voltage gated calcium channels mediate excitatory synaptic transmission in the anterior

MOLECULAR PAINKang et al. Molecular Pain 2013, 9:58http://www.molecularpain.com/content/9/1/58

RESEARCH Open Access

N-type voltage gated calcium channels mediateexcitatory synaptic transmission in the anteriorcingulate cortex of adult miceSukJae Joshua Kang1†, Ming-Gang Liu1,2†, Tian-Yao Shi2,3, Ming-Gao Zhao3, Bong-Kiun Kaang1,4 and Min Zhuo1,2,5*

Abstract

Voltage gated calcium channels (VGCCs) are well known for its importance in synaptic transmission in the peripheraland central nervous system. However, the role of different VGCCs in the anterior cingulate cortex (ACC) has not beenstudied. Here, we use a multi-electrode array recording system (MED64) to study the contribution of different types ofcalcium channels in glutamatergic excitatory synaptic transmission in the ACC. We found that only the N-type calciumchannel blocker ω-conotoxin-GVIA (ω-Ctx-GVIA) produced a great inhibition of basal synaptic transmission, especially inthe superficial layer. Other calcium channel blockers that act on L-, P/Q-, R-, and T-type had no effect. We also testedthe effects of several neuromodulators with or without ω-Ctx-GVIA. We found that N-type VGCC contributed partiallyto (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid- and (R)-Baclofen-induced synaptic inhibition. By contrast, theinhibitory effects of 2-Chloroadenosine and carbamoylcholine chloride did not differ with or without ω-Ctx-GVIA,indicating that they may act through other mechanisms. Our results provide strong evidence that N-type VGCCsmediate fast synaptic transmission in the ACC.

BackgroundIt is well-known that voltage-gated Ca2+ channels (VGCCs)play pivotal roles in neurotransmitter release and synaptictransmission. Previous studies have discovered the role ofvarious types of calcium channels in peripheral regions [1],spinal cord [2], cerebellum [3] and hippocampus [4-6].These studies indicate that N- (CaV2.2) and P/Q-type(CaV2.1) VGCCs play the most dominant role in basal syn-aptic transmission in most of the neurons [7,8]. N-type ismore important in the peripheral nervous system and thejoint action of N- and P/Q-type is prominent in the cen-tral nervous system [9,10]. These studies were per-formed by using the ω-conotoxin GVIA (ω-Ctx GVIA) andω-agatoxin IVA (ω-Aga IVA), which specifically block theN- and P/Q-type VGCCs, respectively.Due to its important role in neuronal Ca2+ concentra-

tion regulation, VGCCs are crucial players in a range of

* Correspondence: [email protected]†Equal contributors1Department of Brain and Cognitive Sciences, College of Natural Sciences,Seoul National University, Seoul 151-746, South Korea2Department of Physiology, Faculty of Medicine, University of Toronto,1 King's College Circle, Toronto, Ontario M5S 1A8, CanadaFull list of author information is available at the end of the article

© 2013 Kang et al.; licensee BioMed Central LtCommons Attribution License (http://creativecreproduction in any medium, provided the orwaiver (http://creativecommons.org/publicdomstated.

physiological and pathological conditions including acutenociception and chronic pain [11-13]. Among differentVGCCs, N- and T- type VGCCs are known to playmajor roles in pain information processing [14,15] andinhibiting VGCCs is thought to be useful for reducingpain [16-18]. Ziconotide (SNX111; Prialt), a drug that tar-gets N-type VGCC approved by the US Food and DrugAdministration and European Medicine Agency, is alsoused intrathecally for severe chronic pain patients [18-21].However, fewer studies have been reported for the roleof VGCCs in synaptic transmission in pain-related cor-tical structures.Convergent evidences from human and animal studies

show that neurons in the anterior cingulate cortex (ACC)play important roles in pain perception and chronic pain[22,23]. Our previous studies show that neuropathic painmodels induced long-term changes in excitatory synaptictransmission in the ACC neurons of adult mice [24,25].Inactivation of the frontal cortex, including the ACC,by local lesions leads to the reduction of the nocicep-tive responses and aversive behaviors associated withchronic pain [26-29]. In situ hybridization brain atlasfrom the Allen Institute for Brain Science shows that

d. This is an Open Access article distributed under the terms of the Creativeommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andiginal work is properly cited. The Creative Commons Public Domain Dedicationain/zero/1.0/) applies to the data made available in this article, unless otherwise

mailto:[email protected]

http://creativecommons.org/licenses/by/2.0

http://creativecommons.org/publicdomain/zero/1.0/

Kang et al. Molecular Pain 2013, 9:58 Page 2 of 13http://www.molecularpain.com/content/9/1/58

N-, P/Q-, L-, T-, and R-type VGCCs are all expressedin the mouse ACC. Thus, in the present study, we useda 64-channel multi-electrode dish (MED64) system, atwo-dimensional electrical activity monitoring device[30-32], to characterize the role of different VGCCs inadult mouse ACC glutamatergic synaptic transmission.The MED64 system allowed us to detect the field exci-tatory postsynaptic potentials (fEPSPs) at multiple sitesin the mouse ACC at the same time, which is difficultto achieve with conventional field recording systems[24,30]. We found that N-type VGCCs play the dominantrole in the ACC synaptic transmission and other VGCCssuch as P/Q-, L-, T-, and R-type do not play any importantrole. Moreover, excitatory synaptic transmission in theACC is subjected to strong and elegant modulation byvarious neuromodulators.

Materials and methodsAnimalsAdult (8–12 week old) male C57BL/6 mice were used.All animals were housed under a 12 h light/dark cyclewith food and water provided ad libitum. All works wereconducted according to the policy and regulation for thecare and use of laboratory animals approved by Institu-tional Animal Care and Use Committee in Seoul NationalUniversity and University of Toronto. The number of ani-mals used and their suffering were greatly minimized.

Brain slice preparationThe general procedures for making ACC slices are simi-lar to those described previously [30]. Briefly, adult malemice were anesthetized with isoflorane and the brainswere removed and transferred to ice-cold artificial cere-brospinal fluid (ACSF) containing (in mM): 124 NaCl,2.5 KCl, 2 CaCl2, 2 MgSO4, 25 NaHCO3, 1 NaH2PO4, 10glucose. This ACSF was used throughout the experiment.Three coronal brain slices (300 μm), after the corpus cal-losum meets, were cut using a vibratome (Leica, Hesse,Germany). The slices were placed in a submerged recov-ery chamber with oxygenated (95% O2, 5% CO2) ACSF atroom temperature for at least 2 h.

Preparation of the multi-electrode arrayThe procedures for preparation of the MED64 system(Panasonic, Osaka, Japan) were similar to those as previ-ously described [30-33]. The MED64 probe (MED-P515A,8 x 8 array, interpolar distance 150 μm, Panasonic)was superfused with ACSF (pH = 7.4) at 28–30°C, andmaintained at a 1 ~ 2 ml/min flow rate. One planar mi-croelectrode with bipolar constant current pulses (1–20 μA, 0.2 ms) was used for stimulation of the ACCslice. The stimulation site was selected within the layerV region. Before use, the surface of the MED64 probewas treated with 0.1% polyethyleneimine (Sigma, St.

Louis, MO, USA) in 25 mM borate buffer (pH 8.4) over-night, at room temperature.

Field potential recording in adult ACC slicesAfter 2 h recovery, one ACC slice was placed in a MEDprobe, and most of the 64 electrodes located within theACC. The slice was allowed to recover for 30 min beforethe electrophysiological recording was attempted. Elec-trical stimulation was delivered to one channel locatedwithin the layer V of the ACC, and evoked fEPSPs weremonitored and recorded from the other 63 channels asdescribed previously [24,30]. The intensity of the stimuliwas approximately 40 ~ 60% of the intensity that inducedthe maximal fEPSPs. Baseline responses were evoked at0.017 Hz for 30 min. The data were averaged every 2 min.

Whole-cell patch-clamp electrophysiologyFor whole-cell patch-clamp electrophysiology, slices wereindividually transferred to a recording chamber on thestage of a BX51WI microscope (Olympus, Tokyo, Japan)equipped with infrared differential interference contrastoptics and superfused with the same ACSF at 2 ml/minfor visualized whole-cell patch-clamp recordings [34-37].Excitatory postsynaptic currents (EPSCs) were recordedfrom superficial layer (layer II-III) pyramidal neurons withan Axon 200B amplifier (Axon Instruments, Union City,CA, USA) and the stimulations were delivered by a bipolartungsten-stimulating electrode placed in the deep layer(layer V-VI). AMPA receptor-mediated EPSCs were in-duced by repetitive stimulations at 0.03 Hz, and neuronswere voltage clamped at −70 mV. The recording pipettes(borosilicate glass, 3–5 MΩ) were filled with a solutioncontaining (in mM) 145 K-gluconate, 5 NaCl, 1 MgCl2,0.2EGTA, 2 Mg-ATP, 0.1 Na3-GTP, 10 HEPES (adjusted topH 7.2 with KOH; 280–300 mOsm). The initial access re-sistance (typically 15–30 MΩ) was monitored throughoutthe experiment. Data were discarded if the access resist-ance changed >15% during an experiment. Data were fil-tered at 1 kHz, and digitized at 10 kHz.

DrugsDrugs were freshly prepared: N-type VGCC blocker ω-CtxGVIA, P/Q-type VGCC blocker ω-Aga IVA and R-typeVGCC blocker SNX-482 were purchased from PeptideInstitute Inc. (Osaka, Japan). T-type VGCC blocker NiCl2was obtained from Sigma. L-type VGCC blocker nimodi-pine, metabotropic glutamate receptor (mGluR) agonist(1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD), cholinergic receptor agonist carbamoylcholinechloride (carbachol, Cch), γ-Aminobutyric acid B (GABAB)receptor agonist (R)-Baclofen and adenosine receptoragonist 2-Chloroadenosine (2-CA) were purchased fromTocris Bioscience (Bristol, UK). Drugs were stored infrozen aliquots at −20°C. All drugs were diluted from the


stock solutions to the final desired concentration in theACSF before immediate use.

Data analysisMED64 Mobius was used for data acquisition and ana-lysis. The percentages of the fEPSP slopes were normal-ized by the averaged value of the baseline period(30 min). The reduction index is calculated as follows:100% - the percentage change of fEPSP slope within thelast 4 min of the experiment. The value used for ‘end ofdrug treatment’ was the averaged value of interval between4 min before and 2 min after endpoint. Whole-cell patch-clamp data were collected and analyzed using Clampex10.2 and Clampfit10.2 software (Axon Instruments). Alldata are presented as mean ± SEM. Statistical comparisonswere made using the t-test and one-way ANOVA bySigmaPlot 11.0. Post hoc Bonferroni test was used for fur-ther comparison. If the data did not pass the equal vari-ance test, one way ANOVA was done in ranks and Dunn’smethod was used for post-hoc test. Two-way ANOVA andPost hoc Bonferroni test was performed in the neuromo-dulator experiment to compare layer difference. In allcases, statistical significance was accepted at the P < 0.05significance level.

ResultsN-type VGCCs in ACC excitatory synaptic transmissionIn the present study, a multi-channel recording system,MED64, was used throughout the experiment to sortout the layer-related differences in synaptic responseswithin the ACC of adult mice. An ACC slice was placedon top of the 8 × 8 square shaped MED64 probe aspreviously reported [24,30]. We stimulated one channelin the deep layer V and observed widespread responsesthroughout the layers except layer I (Figure 1A). In thesuperficial layer, especially layer II/III, most of the chan-nels showed a great reduction after bath application ofthe N-type VGCC blocker, ω-Ctx GVIA (1 μM, 15 min).For example, the two channels within the superficiallayer (Ch. 22 and 38) both underwent similar amountof reduction (39.2% of baseline and 38.2% of baseline,Figure 1B). The effect of the drug was irreversible, withthe reduction not recovered until 1 h after the onset ofthe drug application. The averaged fEPSP slope of 5 acti-vated channels in the superficial layer is similar to the re-sult of one single channel (Figure 1C). Thus, there are noapparent differences among the channels within the samelayer. The summarized data show that the fEPSP slopewas inhibited to 45.8 ± 4.4% of baseline after applying ω-Ctx GVIA (n = 7 slices/7 mice; Figure 1D).To determine if the contribution of the N-type cal-

cium channel to synaptic transmission varies accordingto the cortical layers, we compared the effect of ω-CtxGVIA in different layers in the same slice. We selected

two channels (Ch. 30 and 28) that had similar responsesize and shape in superficial and deep layer (layer V/VI)of the ACC, respectively (Figure 2A). Significant differencewas detected in the reduction process and the inhibitedlevel between the two channels after drug treatment (Ch.30: 37.2% of baseline; Ch. 28: 51.2% of baseline; Figure 2B).The averaged data of 5–7 channels for one single slice(Figure 2C) and the pooled data of several mice demon-strate the same layer-related difference (superficial layer:45.8 ± 4.4% of baseline, deep layer: 59.6 ± 2.8% of baseline,n = 7 slices/7 mice, t(12) = −2.663, P = 0.021; Figure 2D).These results suggest that N-type VGCCs mediate excita-tory synaptic transmission in the ACC and there were dif-ferences between layers.

Role of other VGCCs in ACC synaptic transmissionWe next tested the effects of other VGCC blockers inACC glutamatergic synaptic transmission in the superficiallayer. The P/Q-type VGCC blocker ω-Aga IVA (1 μM,20 min) had no effect at all (Figure 3A). The level of re-sponse was 103.0 ± 1.4% at the end of the drug treatmentand 99.5 ± 1.8% at the end of the recording (n = 5 slices/5mice; Figure 3B). This is different from previous reports ofthe predominant role of P/Q-type VGCC in excitatory syn-aptic transmission in the hippocampus [2,4,6] and cerebel-lum [3]. R-type VGCC has been suggested to contribute toexcitatory synaptic transmission in the rat hippocampus[38]. We addressed the involvement of R-type calciumchannel by bath applying SNX-482 (0.5 μM, 20 min). Wefound that SNX-482 did not produce any significant effect(end of drug: 96.7 ± 1.4%, end of experiment: 97.5 ± 0.8%,n = 6 slices/6 mice; Figure 3C and D). Next, we testedthe possible contribution of low-voltage activated T-typeVGCC by applying the T-type VGCC blocker NiCl2(100 μM, 20 min). We found that there was no change ofthe synaptic transmission in the superficial layer (end ofdrug: 98.8 ± 4.4%, end of experiment: 97.8 ± 2.2%, n = 7slices/7 mice; Figure 3E and F). Finally, we tested if theL-type VGCC mediates the ACC synaptic transmission.Bath infusion of nimodipine (10 μM, 20 min) failed toproduce any inhibitory effect on the slope of fEPSP re-corded from the superficial layer of the ACC slice (endof drug: 101.2 ± 3.3%, end of experiment: 99.2 ± 2.3%, n = 5slices/5 mice; Figure 3G and H).Similar results were found in the deep layer of the

ACC. The ω-Aga IVA group showed 105.2 ± 1.4% at theend of drug treatment and 96.9 ± 1.4% at the end of experi-ment (n = 5 slices/5 mice; Figure 4A and B). SNX-482 (endof drug: 104.1 ± 0.7%, end of experiment: 98.0 ± 0.2%, n = 6slices/6 mice; Figure 4C and D), NiCl2 (end of drug: 91.2 ±3.2%, end of experiment: 97.7 ± 1.3%, n = 7 slices/7 mice;Figure 4E and F) and nimodipine (end of drug: 99.3 ±2.1%, end of experiment: 98.0 ± 1.9%, n = 5 slices/5 mice;Figure 4G and H) all showed no inhibition.

Figure 2 Comparison of the effects of the N-type VGCC blocker in the superficial layer and deep layer of the ACC. A, The same slice asFigure 1A. Two channels (Ch. 30 from LII/III and Ch. 28 from LV/VI) are selected for further comparison. B, ω-Ctx GVIA (1 μM) was bath applied for15 min (filled circle: Ch. 30; open circle: Ch. 28) in a single slice. Sample fEPSP recordings taken at the times indicated by the corresponding numbersare shown above the plot. Calibration: 500 μV, 20 ms. C, Averaged data of 5–7 activated channels in the superficial layer (filled circle: LII/III) and deeplayer (open circle: LV/VI) in one slice. D, Pooled data of 7 mice (LII/III: 45.8 ± 4.4% of baseline, LV/VI: 59.6 ± 2.8% of baseline, n = 7 slices/7 mice,t(12) = −2.663, P = 0.021). ω-Ctx GVIA produced a stronger inhibition of superficial layer responses than those of the deep layer in the ACC. Thehorizontal bars indicate the period of drug application. Asterisk indicates the statistical significance between superficial layer and deep layer of theACC slice. Error bars represent SEM.

Figure 1 Role of N-type VGCCs in ACC excitatory synaptic transmission in the superficial layer. A, A representative example of MED64responses before (black) and after (red) ω-Ctx GVIA (1 μM) application. Stimulation (thunderbolt) was given in the deep layer (Ch. 36). The regionsare divided into Layer (L) I, II/III and V/VI (vertical lines). Two channels (Ch. 22 and Ch. 38) are selected for further comparison. B, ω-Ctx GVIA(1 μM) was applied for 15 min (filled circle: Ch. 22; open circle: Ch. 38) in a single slice. Sample fEPSP recordings taken at the times indicated bythe corresponding numbers are shown above the plot. Calibration: 300 μV, 20 ms. C, Averaged data of 5 activated channels in the superficial layerin one slice. D, Pooled data of 7 mice (45.8 ± 4.4% of baseline, n = 7 slices/7 mice). Application of ω-Ctx GVIA produced an irreversible inhibitionof synaptic responses recorded from the superficial layer of the ACC. The horizontal bars indicate the period of drug application. Error barsrepresent SEM.


Figure 3 Role of other VGCCs in ACC excitatory synaptic transmission in the superficial layer. A, Averaged data of 5 channels in the superficiallayer in one slice, showing the effect of applying ω-Aga IVA (1 μM) for 20 min. B, Pooled data of 5 mice (99.5 ± 1.8% of baseline, n = 5 slices/5 mice).C, Averaged data of 6 channels in the superficial layer in one slice, showing the effect of applying SNX-482 (0.5 μM) for 20 min. D, Pooled data of 6mice (97.5 ± 0.8% of baseline, n = 6 slices/6 mice). E, Averaged data of 5 channels in the superficial layer in one slice, showing the effect of applyingNiCl2 (100 μM) for 20 min. F, Pooled data of 7 mice (97.8 ± 2.2% of baseline, n = 7 slices/7 mice). G, Averaged data of 5 channels in the superficial layerin one slice, showing the effect of applying nimodipine (10 μM) for 20 min. H, Pooled data of 5 mice (99.2 ± 2.3% of baseline, n = 5 slices/5 mice). Alldrugs infused had no effect on synaptic transmission in the superficial layer of the ACC. The horizontal bars indicate the period of drug application.Error bars represent SEM.


The difference in the reduction index among the drugtreatment groups in the superficial layer was statisticallysignificant (F(4,25) = 16.00, P = 0.003, One-way ANOVAin ranks with Dunn’s post-hoc; P < 0.05 between N-typeand all other groups, Figure 5A). There was also signifi-cant difference in the deep layer (F(4,25) = 93.53, P < 0.001,One-way ANOVA with Bonferroni’s post-hoc; P < 0.001between N-type and all other groups, Figure 5B). Overall,N-type is the predominant VGCC involved in excitatorysynaptic transmission in the ACC. Other VGCCs wetested do not participate in the ACC glutamatergic synap-tic transmission.

Modulation of ACC synaptic transmissionWe next investigated whether the ACC synaptic trans-mission is responsive to the modulation exerted by sev-eral neuromodulators in the brain. Neuromodulatorsare signals that trigger activation or inhibition of the

neurons and many of them are known to affect VGCCs[4,39-41]. Because only the N-type VGCC blocker showeda significant reduction in the ACC synaptic transmis-sion, we decided to apply several neuromodulators withor without the presence of ω-Ctx GVIA (1 μM). 1S,3R–ACPD (200 μM, 10 min), an mGluR agonist, was firsttested without applying ω-Ctx GVIA (Figure 6A). Onechannel in the superficial layer (Ch. 19) and the other inthe deep layer (Ch. 38) were selected to compare its ef-fect. The drug induced a great inhibition during the treat-ment and after washout the response recovered to thebaseline level in both superficial and deep layers. The re-duction extent of deep layer during drug application wasless than that of the superficial layer (Ch. 19: 22.0% ofbaseline; Ch. 38: 44.6% of baseline, Figure 6B). This trendwas also seen in the averaged data of 5–7 channels in oneslice (Figure 6C). Pooled data from 7 animals show thesame layer-related difference (LII/III: 23.2 ± 6.5%, LV/VI:

Figure 4 Role of other VGCCs in ACC excitatory synaptic transmission in the deep layer. A, Averaged data of 6 channels in the deep layer in oneslice, showing the effect of applying ω-Aga IVA (1 μM) for 20 min. B, Pooled data of 5 mice (96.9 ± 1.4% of baseline, n = 5 slices/5 mice). C, Averaged dataof 8 channels in the deep layer in one slice, showing the effect of applying SNX-482 (0.5 μM) for 20 min. D, Pooled data of 6 mice (98.0 ± 0.2% of baseline,n = 6 slices/6 mice). E, Averaged data of 6 channels in the deep layer in one slice, showing the effect of applying NiCl2 (100 μM) for 20 min. F, Pooleddata of 7 mice (97.7 ± 1.3% of baseline, n = 7 slices/7 mice). G, Averaged data of 7 channels in the deep layer in one slice, showing the effect of applyingnimodipine (10 μM) for 20 min. H, Pooled data of 5 mice (98.0 ± 1.9% of baseline, n = 5 slices/5 mice). All drugs infused had no effect on synaptictransmission in the deep layer of the ACC. The horizontal bars indicate the period of drug application. Error bars represent SEM.


42.7 ± 5.5%, n = 7 slices/7 mice, t(8) = −2.280, P = 0.042;Figure 6D).To compare the inhibitory effects among different

neuromodulators, we sequentially applied several drugsto the same slice. After the application of 1S,3R–ACPD(200 μM) for 10 min, we then applied GABAB receptoragonist (R)-Baclofen (5 μM) for 5 min, adenosine recep-tor agonist 2-CA (5 μM) for 5 min and finally choliner-gic receptor agonist carbachol (Cch, 10 μM) for 10 min(Figure 6E). In a previous study of the hippocampus,these drugs all inhibited the synaptic transmission im-mediately after they were applied with full recovery afterwashout [4]. We found similar results in the ACC slicesbut the recovery was partial after the washout. In thesuperficial layer, 1S,3R–ACPD inhibited the response to25.7 ± 3.9% of baseline, (R)-Baclofen to 19.7 ± 2.8%, 2-CAto 49.4 ± 5.5%, and Cch to 38.5 ± 4.2% (n = 5 slices/5 mice).The magnitude of inhibition induced by the drugs in thedeep layer was: 1S,3R–ACPD 47.5 ± 2.3%, (R)-Baclofen

48.8 ± 2.2%, 2-CA 52.9 ± 5.2% and Cch 42.6 ± 2.5% (n = 5slices/5 mice). Generally, there was statistically significantdifference between layers (F(1,8) = 6.427, P = 0.035, Re-peated two-way ANOVA). Post hoc Bonferroni analysisrevealed statistical differences between LII/III and LV/VIfor 1S,3R–ACPD (P = 0.013) and (R)-Baclofen (P < 0.001).2-CA (P = 0.673) and Cch (P = 0.714) showed no layer-related difference (see Figure 6E). Analysis of the reduc-tion index obtained the same results (Figure 6F). Both1S,3R–ACPD and (R)-Baclofen produced a much strongerinhibition in the superficial layer than in the deep layer(P < 0.001 for both drugs).We next checked whether these effects will change

after applying ω-Ctx GVIA (1 μM, 15 min) to block N-type VGCC-mediated synaptic transmission (Figure 7A-C).Similarly, ω-Ctx GVIA induced irreversible synaptic inhib-ition even after washout. We then applied the neuromodu-lator as Figure 6E. All the drugs still produced inhibition ofthe fEPSP recorded from the ACC. The inhibited values in

Figure 5 The predominant role of N-type VGCC in ACC excitatorysynaptic transmission. A, The difference in the reduction index(100% - the normalized fEPSP slope value of the last 4 min of theexperiment) among the drug treatment groups in the superficial layeris statistically significant (F(4,25) = 16.00, P = 0.003, One-way ANOVA inranks with Dunn’s post-hoc; P < 0.05 between N-type and all othergroups). B, The difference in the deep layer is also statisticallysignificant (F(4,25) = 93.53, P < 0.001, One-way ANOVA with Bonferroni’spost-hoc; P < 0.001 between N-type and all other groups). Error barsrepresent SEM.


the superficial layer were: 1S,3R–ACPD 16.7 ± 2.4%, (R)-Baclofen 14.8 ± 0.3%, 2-CA 19.2 ± 1.9%, Cch 14.0 ± 0.7%(n = 5 slices/5 mice, Figure 7C). In the deep layer, thevalues were: 1S,3R–ACPD 12.3 ± 3.7%, (R)-Baclofen 14.4 ±4.8%, 2-CA 22.8 ± 1.1%, Cch 18.5 ± 2.1% (n = 5 slices/5mice, Figure 7C). Repeated two-way ANOVA detected thestatistically significant differences between layers (F(1,8) =7.069, P = 0029). However, post doc Bonferroni test failedto show any layer-related significant change for all of thefour drugs (Figure 7D). Thus, although the N-type VGCCblocker did not prevent neuromodulators-induced synapticinhibition, it abolished the layer difference in the effects of1S,3R–ACPD and (R)-Baclofen.

To further elucidate the mechanisms for this loss oflayer difference, we normalized the 70–90 min period ofFigure 7C as 100% and checked the reduction index of theneuromodulators again (Figure 7E). We found that 1S,3R–ACPD inhibited the ω-Ctx GVIA-insensitive responsesfrom the deep layer to a greater extent than those in thesuperficial layer (LII/III: 54.6 ± 7.0%, LV/VI: 77.7 ± 5.0%,P = 0.007). The layer difference was not clear for Baclofen(LII/III: 59.3 ± 2.9%, LV/VI: 75.0 ± 6.3%, P = 0.057), 2-CA(LII/III: 46.3 ± 8.3%, LV/VI: 55.4 ± 6.5%, P = 0.262) andCch (LII/III: 61.9 ± 1.5%, LV/VI: 64.8 ± 4.3%, P = 0.719,Figure 7E). Comparison of the reduction index with andwithout the presence of ω-Ctx GVIA reached the sameconclusion for the layer difference (Figure 7F and G).These data suggest that 1S,3R–ACPD possibly acts morein the deep layer with different mechanisms when theN-type VGCC was blocked.

Whole-cell patch-clamp recordings of synaptic transmissionin the ACCTo confirm the MED64 data, we performed whole-cell patch-clamp recordings in the ACC LII/III neu-rons (Figure 8). Three VGCC blockers were bathinfused for 30 min and only ω-Ctx GVIA showed theblocking effect in the ACC synaptic transmission (ω-Ctx GVIA: 39.2 ± 7.0% of baseline, n = 10 cells/6 mice;nimodipine: 101.2 ± 3.0% of baseline, n = 10 cells/6mice; NiCl2: 99.6 ± 5.1% of baseline, n = 10 cells/6mice; Figure 8A-C). In addition, we also applied theneuromodulators and examined their influence on theEPSCs recorded from the superficial layer of theACC. Similarly, we found that all of the four drugsproduced an acute inhibition that can be fully or par-tially recovered after washout (1S,3R–ACPD: 200 μM,29.1 ± 5.1% of baseline; Cch: 10 μM, 46.7 ± 6.2% ofbaseline; (R)-Baclofen: 5 μM, 41.7 ± 2.0% of baseline;2-CA: 5 μM, 29.9 ± 5.3% of baseline; n = 8 cells/4mice; Figure 8D-G). Nevertheless, comparison of thewhole-cell and MED64 data revealed some differencesin the reduction extent and recover process of thedrug effect. For example, 2-CA produced a much lar-ger extent of reduction in EPSCs (29.9 ± 5.3% of base-line at the end of the drug treatment, n = 8 cells/4mice, Figure 8G) as compared to its effect on fEPSPs(49.4 ± 5.5% of baseline at the end of the drug treat-ment, n = 5 slices/5 mice, Figure 6E). Also, while the 2-CA-induced fEPSP inhibition is fully reversible (Figure 6E),the suppression of EPSCs cannot be recovered (Figure 8G).This discrepancy may be attributed to differences inexperimental variables such as the recording method(whole-cell patch-clamp recording vs. multi-channelfield potential recording) and drug infusion time(30 min vs. 5 min).

Figure 6 Effect of neuromodulators on ACC excitatory synaptic transmission. A, A representative example of MED64 responses before (black)and after (red) 1S,3R–ACPD (200 μM) application. Stimulation (thunderbolt) was given in the deep layer (Ch. 37). Two channels (Ch. 19 from LII/III andCh. 38 from LV/VI) are selected for further comparison. B, 1S,3R–ACPD (200 μM) was bath applied for 10 min (filled circle: Ch. 19; open circle: Ch. 38) ina single slice. Sample fEPSP recordings taken at the times indicated by the corresponding numbers are shown above the plot. Calibration: 300 μV,20 ms. C, Averaged data of 5–7 activated channels in the superficial layer and deep layer in one slice. D, Pooled data of 7 mice (LII/III: 23.2 ± 6.5% ofbaseline, LV/VI: 42.7 ± 5.5% of baseline, n = 7 slices/7 mice). Bath application of 1S,3R-ACPD resulted in an acute inhibition that gradually recovered afterwashout. The superficial layer response exhibited a much more reduction than the deep layer. E, Four neuromodulators were sequentially applied.Significant differences could be found in the blocking effect of 1S,3R–ACPD and (R)-Baclofen between individual layers (n = 5 slices/5 mice). Thehorizontal bars indicate the period of drug application. Error bars represent SEM. F, Reduction index of the neuromodulators. 1S,3R–ACPD (LII/III:74.3% ± 8.8%, LV/VI: 52.5 ± 2.3%, P = 0.003) and (R)-Baclofen (LII/III: 80.3 ± 2.8%, LV/VI: 51.1 ± 2.2%, P = 0.002) showed statistically significant differencebetween the layers. 2-CA (LII/III: 50.6 ± 5.5%, LV/VI: 47.1 ± 5.2%, P = 0.614) and Cch (LII/III: 61.5 ± 4.2%, LV/VI: 57.3 ± 2.5%, P = 0.538) exhibited nolayer-related difference.


DiscussionIn this study, we have demonstrated the prominent roleof N-type VGCC in mediating glutamatergic synaptictransmission in the adult mice ACC. We have usedthe newly-developed MED64 system to record multi-site synaptic responses in the coronal ACC slices andcompare the possible layer-related differences. Severaltypes of VGCC blockers were tested in the basal syn-aptic transmission and only ω-Ctx GVIA, the N-typeVGCC blocker, showed a great reduction. Moreover, thesuperficial layer had a greater inhibition than the deeplayer. We also tested whether ω-Ctx GVIA would influ-ence the modulatory effect of several neuromodulatorson excitatory synaptic transmission in the ACC. Wefound that the neuromodulator effects were not greatlyaffected by ω-Ctx GVIA.

N- and P/Q-type VGCC in the ACCN- and P/Q-type calcium channels are the major VGCCsfor Ca2+ influx to initiate the fast release of neurotrans-mitters/neuromodulators such as glutamate, acetylcholine,and GABA [42]. Both channels are high-voltage activatedchannels consisting of the α1 subunit pore. N-type VGCCcomprises an α1B subunit or Cav2.2 and the channel is pri-marily in the presynaptic compartment [8,18,42]. ω-CtxGVIA was commonly used to antagonize the activity ofN-type VGCC [1,4,6,18,20]. There are studies demon-strating the up-regulation of N-type VGCC subunit inthe primary afferent neurons after tissue inflammationand nerve injury [12,43,44]. There is an N-type VGCC-targeting drug (Ziconotide) used in the clinic to relieveneuropathic and inflammation pain. However, its clinicalapplication is limited by the accompanying central side

Figure 7 Effect of neuromodulators after blocking N-type VGCC. A, Results of one channel applying each drug sequentially (filled circle: LII/III, opencircle: LV/VI): ω-Ctx-GVIA (1 μM) for 15 min, 1S,3R–ACPD (200 μM) for 10 min, (R)-Baclofen (5 μM) for 5 min, 2-CA (5 μM) for 5 min and Cch (10 μM) for10 min. B, Averaged data (n = 6–8 channels/1 slice) of each layer that showed response in one slice. C, Pooled data of 5 mice. Pre-exposure of the ACCslice with ω-Ctx-GVIA abolished the layer-related difference in the effect of 1S,3R–ACPD and (R)-Baclofen (n = 5 slices/5 mice). The horizontal bars indicatethe period of drug application. Error bars represent SEM. D, Reduction index of ω-Ctx-GVIA and neuromodulators in different layers. All drugs showed nostatistically significant difference between layers. E, Reduction index of neuromodulators when ω-Ctx-GVIA-insensitive responses (70–90 min in Figure 7C)were normalized as 100%. 1S,3R–ACPD inhibited the ω-Ctx GVIA-insensitive responses more in the deep layer (LII/III: 54.6 ± 7.0%, LV/VI: 77.7 ± 5.0%,P= 0.007). The layer difference was not clear for Baclofen (LII/III: 59.3 ± 2.9%, LV/VI: 75.0 ± 6.3%, P = 0.057), 2-CA (LII/III: 46.3 ± 8.3%, LV/VI: 55.4 ± 6.5%,P= 0.262) and Cch (LII/III: 61.9 ± 1.5%, LV/VI: 64.8 ± 4.3%, P = 0.719). F, Reduction index of each agonist in the superficial layer with (Figure 6F) orwithout ω-Ctx GVIA (Figure 7E). G, Reduction index of each agonist in the deep layer with (Figure 6F) or without ω-Ctx GVIA (Figure 7E).


effects and can only be applied spinally [18-21]. Our stud-ies provide strong evidence that N-type calcium channelsplay important roles in the ACC synaptic transmission,and the inhibitory effect of the N-type VGCC blocker inthe ACC may explain some of the side effects caused byZiconotide.P/Q-type VGCC consists of an α1A subunit or Cav2.1

and ω-Aga IVA is the well-known antagonist. Several stud-ies show that dysfunction of this channel induces ataxia,migraine, vertigo and epilepsy [45-48]. The involvement of

P/Q-type VGCC in central synaptic transmission and plas-ticity has been extensively studied in the hippocampus[2,4,6], cerebellum [3] and nucleus accumbens [49]. How-ever, there are no studies of N- and P/Q-type VGCC en-gagement in the adult mice ACC. Our results revealed adramatic reduction of glutamatergic synaptic transmissionby the N-type VGCC blocker in the ACC. Since the excita-tory synaptic responses in the ACC are mediated predom-inantly by AMPA/kainate receptors, the explanation forω-Ctx GVIA-induced inhibition would be the reduction

Figure 8 Whole-cell patch-clamp recordings of the synaptic responses in the ACC: N-type VGCC involvement and modulation byneuromodulators. A, ω-Ctx-GVIA (1 μM, 30 min) treatment blocked the EPSCs recorded from LII/III neurons of the ACC (39.2 ± 7.0%, n = 10cells/6 mice). Sample traces are shown in the insets. B, C, Nimodipine (10 μM, 30 min, 101.2 ± 3.0%, n = 10 cells/6 mice) and NiCl2 (100 μM,30 min, 99.6 ± 5.1%, n = 10 cells/6 mice) did not block the synaptic response. Sample traces of nimodipine treatment are shown in the insets.D-F, Application of 1S,3R–ACPD (200 μM, 29.1 ± 5.1%, n = 8 cells/4 mice), Cch (10 μM, 46.7 ± 6.2%, n = 8 cells/4 mice) and (R)-Baclofen (5 μM,41.7 ± 2.0%, n = 8 cells/4 mice) for 30 min inhibited the ACC LII/III neuronal responses and the inhibition was recovered after washout. G, 2-CA (5 μM,29.9 ± 5.3%, n = 8 cells/4 mice) induced a similar acute inhibition but the inhibition is maintained during washout. The horizontal bars indicate theperiod of drug application. Error bars represent SEM.


of presynaptic glutamate release [30]. Interestingly, ω-Ctx GVIA produced a much stronger inhibition of thesuperficial layer responses than those of the deep layer,indicating that the expression density of the N-typeVGCC in the ACC may have layer-related difference. Fu-ture studies are required to address this issue by usingmorphological tools to investigate the distribution ofVGCCs across each layer of the ACC.The current findings show other VGCC blockers did not

significantly inhibit ACC synaptic response. These results

differ from previous reports in other brain regions, whichinvolve the combination of N- and P/Q-type VGCCs inexcitatory synaptic transmission [9,10]. The possibilityof low doses of the drugs causing the negative results isunlikely, because the drug concentrations used in thepresent experiment are not lower compared to otherstudies [4,10,49]. According to the in situ hybridizationdata from Allen Institute for Brain Sciences, all types ofVGCCs are expressed in the ACC. Therefore, they mayexert some other functions rather than mediating the


excitatory synaptic transmission. For example, it has beenshown in our previous studies that L-type VGCC is in-volved in low frequency stimulation-induced long-termdepression in the ACC [30,50]. Taken together, these find-ings indicate that different central synapses in the brainmay depend on different types of VGCCs for mediation ofthe excitatory synaptic transmission. Notably, however,ω-Ctx GVIA only blocked about 50% of the total synaptictransmission in the ACC. Therefore, it is still necessary forfuture studies to identify the receptors or channels mediat-ing the remaining 50% of the ACC synaptic response inthe basal condition.

VGCCs and neuromodulators in the ACCN-, P/Q-, R-, and L- type VGCCs are all involved in pre-synaptic neurotransmitter release [9,42] and it has beenreported that different neuromodulators affect theseVGCCs [4,39-41]. Thus, we wanted to test the effect ofω-Ctx GVIA on the modulation of ACC synaptic trans-mission exerted by various neuromodulators. We haveapplied four neuromodulators with or without the pres-ence of ω-Ctx GVIA. All drugs induced a great acute in-hibition of the fEPSP under both conditions and theinhibitive effect is reversible after washout. These resultsare partially consistent with the previous reports in thehippocampus [4]. Interestingly, the layer-related differ-ence regarding the inhibitory effect of 1S,3R–ACPD and(R)-Baclofen is abolished after blocking N-type VGCC-mediated synaptic transmission. One possible explan-ation for layer-related difference is different distributionof mGluRs (for 1S,3R–ACPD) and GABAB (for (R)-Bac-lofen) among synapses in the superficial vs deep layers ofthe ACC. Future studies are needed to further investigatethese mechanisms via a combination of electrophysio-logical and morphological approaches.The modulation of synaptic transmission by 1S,3R–

ACPD in the ACC is consistent with the previous reportsin other brain areas, such as hippocampus [4,51-53], neo-cortex [54], cerebellum [55] and striatum [56]. These dataare also consistent with the previous results showing the in-hibition of N-type and other types of VGCC currents bythe 1S,3R-ACPD [57-63]. It is likely that 1S,3R-ACPD pro-duces its inhibitory effect by intracellular G protein coupledsignaling pathways. Considering the fact that 1S,3R-ACPDis a non-selective mGluR antagonist [64,65], future studiesare needed to examine the subtypes of mGluRs and theirrelated downstream signaling pathways mediating the in-hibitory effects.In summary, our present study is the first to establish

the importance of N-type VGCC in mediating excitatorysynaptic transmission in the adult mice ACC. ACC isknown to be an important region for memory and chronicpain [22,23]. Due to its important roles in normal synaptictransmission in the ACC, our results provide possible

explanations for the central side effects produced by theN-type calcium channel blockers applied intrathecally orsystemically in the clinic.

Abbreviations1S,3R-ACPD: (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid; 2-CA:2-Chloroadenosine; ACC: Anterior cingulate cortex; ACSF: Artificialcerebrospinal fluid; Cch: Carbachol; EPSC: Excitatory postsynaptic current;fEPSP: Field excitatory postsynaptic potential; GABAB: γ-Aminobutyric acid B;MED64: 64-channel multi-electrode dish; mGluR: Metabotropic glutamatereceptor; VGCC: Voltage-gated calcium channel; ω-Ctx GVIA: ω-conotoxinGVIA; ω-Aga IVA: ω-agatoxin IVA.

Competing interestsThe authors declare that they have no competing interests.

Authors’ contributionsSJK designed, performed the experiments and wrote the manuscript. MGLperformed some of the MED64 experiments and revised the manuscript. TYSperformed the whole-cell patch-clamp recordings. MGZ and BKK supervisedthe experiments and commented on the manuscript. MZ participated in theexperiment design and coordination, and helped to finish the manuscript.All authors read and approved the final manuscript.

AcknowledgementsThis work was supported by the World-Class University (WCU) program ofthe Ministry of Education, Science and Technology in Korea through NationalResearch Foundation (R32-10142). M.Z. was supported by CIHR operatinggrant, Canada Research Chair (CRC), NSEC discovery grant 402555, and WCU.B.K.K. is a Yonam Foundation Scholar and supported by WCU and the Na-tional Honor Scientist Program, Korea.

Author details1Department of Brain and Cognitive Sciences, College of Natural Sciences,Seoul National University, Seoul 151-746, South Korea. 2Department ofPhysiology, Faculty of Medicine, University of Toronto, 1 King's CollegeCircle, Toronto, Ontario M5S 1A8, Canada. 3Department ofPharmacology, School of Pharmacy, Fourth Military Medical University,Xi'an 710032, China. 4Department of Biological Sciences, College ofNatural Sciences, National Creative Research Initiative Center forMemory, Seoul National University, Seoul 151-747, South Korea. 5Centerfor Neuron and Disease, Frontier Institute of Science and Technology,Xi’an Jiaotong University, Xi’an 710049, China.

Received: 1 October 2013 Accepted: 4 November 2013Published: 14 November 2013

References1. Wright CE, Angus JA: Effects of N-, P- and Q-type neuronal calcium channel

antagonists on mammalian peripheral neurotransmission. Br J Pharmacol1996, 119(1):49–56.

2. Takahashi T, Momiyama A: Different types of calcium channels mediatecentral synaptic transmission. Nature 1993, 366(6451):156–158.

3. Regehr WG, Mintz IM: Participation of multiple calcium channel types intransmission at single climbing fiber to Purkinje cell synapses.Neuron 1994, 12(3):605–613.

4. Wheeler DB, Randall A, Tsien RW: Roles of N-type and Q-type Ca2+channels in supporting hippocampal synaptic transmission. Science 1994,264(5155):107–111.

5. Castillo PE, Weisskopf MG, Nicoll RA: The role of Ca2+ channels inhippocampal mossy fiber synaptic transmission and long-termpotentiation. Neuron 1994, 12(2):261–269.

6. Luebke JI, Dunlap K, Turner TJ: Multiple calcium channel types controlglutamatergic synaptic transmission in the hippocampus. Neuron 1993,11(5):895–902.

7. Dunlap K, Luebke JI, Turner TJ: Exocytotic Ca2+ channels in mammaliancentral neurons. Trends Neurosci 1995, 18(2):89–98.

8. Olivera BM, Miljanich GP, Ramachandran J, Adams ME: Calcium channeldiversity and neurotransmitter release: the omega-conotoxins andomega-agatoxins. Annu Rev Biochem 1994, 63:823–867.


9. Catterall WA, Few AP: Calcium channel regulation and presynapticplasticity. Neuron 2008, 59(6):882–901.

10. Cao YQ, Tsien RW: Different relationship of N- and P/Q-type Ca2+ channelsto channel-interacting slots in controlling neurotransmission at culturedhippocampal synapses. J Neurosci 2010, 30(13):4536–4546.

11. Gribkoff VK: The role of voltage-gated calcium channels in pain andnociception. Semin Cell Dev Biol 2006, 17(5):555–564.

12. Cao YQ: Voltage-gated calcium channels and pain. Pain 2006,126(1–3):5–9.

13. Park J, Luo ZD: Calcium channel functions in pain processing.Channels (Austin) 2010, 4(6):510–517.

14. Zamponi GW, Lewis RJ, Todorovic SM, Arneric SP, Snutch TP: Role ofvoltage-gated calcium channels in ascending pain pathways. Brain ResRev 2009, 60(1):84–89.

15. Campbell JN, Meyer RA: Mechanisms of neuropathic pain. Neuron 2006,52(1):77–92.

16. Dogrul A, Gardell LR, Ossipov MH, Tulunay FC, Lai J, Porreca F: Reversal ofexperimental neuropathic pain by T-type calcium channel blockers.Pain 2003, 105(1–2):159–168.

17. Brittain JM, Duarte DB, Wilson SM, Zhu W, Ballard C, Johnson PL, Liu N,Xiong W, Ripsch MS, Wang Y, et al: Suppression of inflammatory andneuropathic pain by uncoupling CRMP-2 from the presynaptic Ca2+

channel complex. Nat Med 2011, 17(7):822–829.18. Adams DJ, Callaghan B, Berecki G: Analgesic conotoxins: block and G

protein-coupled receptor modulation of N-type (Ca(V) 2.2) calciumchannels. Br J Pharmacol 2012, 166(2):486–500.

19. Atanassoff PG, Hartmannsgruber MW, Thrasher J, Wermeling D, Longton W,Gaeta R, Singh T, Mayo M, McGuire D, Luther RR: Ziconotide, a new N-typecalcium channel blocker, administered intrathecally for acute postoperativepain. Reg Anesth Pain Med 2000, 25(3):274–278.

20. Perret D, Luo ZD: Targeting voltage-gated calcium channels for neuropathicpain management. Neurotherapeutics 2009, 6(4):679–692.

21. Staats PS, Yearwood T, Charapata SG, Presley RW, Wallace MS, Byas-Smith M,Fisher R, Bryce DA, Mangieri EA, Luther RR, et al: Intrathecal ziconotide inthe treatment of refractory pain in patients with cancer or AIDS: arandomized controlled trial. JAMA 2004, 291(1):63–70.

22. Vogt BA: Pain and emotion interactions in subregions of the cingulategyrus. Nat Rev Neurosci 2005, 6(7):533–544.

23. Zhuo M: Cortical excitation and chronic pain. Trends Neurosci 2008,31(4):199–207.

24. Li XY, Ko HG, Chen T, Descalzi G, Koga K, Wang H, Kim SS, Shang Y, Kwak C,Park SW, et al: Alleviating neuropathic pain hypersensitivity by inhibitingPKMzeta in the anterior cingulate cortex. Science 2010, 330(6009):1400–1404.

25. Xu H, Wu LJ, Wang H, Zhang X, Vadakkan KI, Kim SS, Steenland HW, ZhuoM: Presynaptic and postsynaptic amplifications of neuropathic pain inthe anterior cingulate cortex. J Neurosci 2008, 28(29):7445–7453.

26. Yen CP, Kuan CY, Sheehan J, Kung SS, Wang CC, Liu CK, Kwan AL: Impact ofbilateral anterior cingulotomy on neurocognitive function in patientswith intractable pain. J Clin Neurosci 2009, 16(2):214–219.

27. Yen CP, Kung SS, Su YF, Lin WC, Howng SL, Kwan AL: Stereotacticbilateral anterior cingulotomy for intractable pain. J Clin Neurosci2005, 12(8):886–890.

28. Johansen JP, Fields HL, Manning BH: The affective component of pain inrodents: direct evidence for a contribution of the anterior cingulatecortex. Proc Natl Acad Sci U S A 2001, 98(14):8077–8082.

29. Zhuo M: Molecular mechanisms of pain in the anterior cingulate cortex.J Neurosci Res 2006, 84(5):927–933.

30. Kang SJ, Liu MG, Chen T, Ko HG, Baek GC, Lee HR, Lee K, Collingridge GL,Kaang BK, Zhuo M: Plasticity of metabotropic glutamate receptor-dependentlong-term depression in the anterior cingulate cortex after amputation.J Neurosci 2012, 32(33):11318–11329.

31. Liu MG, Kang SJ, Shi TY, Koga K, Zhang MM, Collingridge GL, Kaang BK,Zhuo M: Long-term potentiation of synaptic transmission in the adultmouse insular cortex: multielectrode array recordings. J Neurophysiol2013, 110(2):505–521.

32. Liu MG, Koga K, Guo YY, Kang SJ, Collingridge GL, Kaang BK, Zhao MG,Zhuo M: Long-term depression of synaptic transmission in the adultmouse insular cortex in vitro. Eur J Neurosci 2013, 38(8):3128–3145.

33. Oka H, Shimono K, Ogawa R, Sugihara H, Taketani M: A new planarmultielectrode array for extracellular recording: application tohippocampal acute slice. J Neurosci Methods 1999, 93(1):61–67.

34. Zhao MG, Ko SW, Wu LJ, Toyoda H, Xu H, Quan J, Li J, Jia Y, Ren M, Xu ZC,et al: Enhanced presynaptic neurotransmitter release in the anteriorcingulate cortex of mice with chronic pain. J Neurosci 2006, 26(35):8923–8930.

35. Toyoda H, Zhao MG, Xu H, Wu LJ, Ren M, Zhuo M: Requirement ofextracellular signal-regulated kinase/mitogen-activated protein kinasefor long-term potentiation in adult mouse anterior cingulate cortex. MolPain 2007, 3:36.

36. Wu LJ, Ko SW, Toyoda H, Zhao MG, Xu H, Vadakkan KI, Ren M, Knifed E,Shum F, Quan J, et al: Increased anxiety-like behavior and enhancedsynaptic efficacy in the amygdala of GluR5 knockout mice. PLoS One 2007,2(1):e167.

37. Wu LJ, Zhang XH, Fukushima H, Zhang F, Wang H, Toyoda H, Li BM, Kida S,Zhuo M: Genetic enhancement of trace fear memory and cingulatepotentiation in mice overexpressing Ca2+/calmodulin-dependent proteinkinase IV. Eur J Neurosci 2008, 27(8):1923–1932.

38. Gasparini S, Kasyanov AM, Pietrobon D, Voronin LL, Cherubini E: PresynapticR-type calcium channels contribute to fast excitatory synaptic transmissionin the rat hippocampus. J Neurosci 2001, 21(22):8715–8721.

39. Gross RA, Macdonald RL, Ryan-Jastrow T: 2-Chloroadenosine reduces the Ncalcium current of cultured mouse sensory neurones in a pertussistoxin-sensitive manner. J Physiol 1989, 411:585–595.

40. Lester RA, Jahr CE: Quisqualate receptor-mediated depression of calciumcurrents in hippocampal neurons. Neuron 1990, 4(5):741–749.

41. Mintz IM, Bean BP: GABAB receptor inhibition of P-type Ca2+ channels incentral neurons. Neuron 1993, 10(5):889–898.

42. Catterall WA: Voltage-gated calcium channels. Cold Spring Harb PerspectBiol 2011, 3(8):a003947.

43. McGivern JG, McDonough SI: Voltage-gated calcium channels as targetsfor the treatment of chronic pain. Curr Drug Targets CNS Neurol Disord2004, 3(6):457–478.

44. Yaksh TL: Calcium channels as therapeutic targets in neuropathic pain.J Pain 2006, 7(1 Suppl 1):S13–30.

45. Jun K, Piedras-Renteria ES, Smith SM, Wheeler DB, Lee SB, Lee TG, Chin H,Adams ME, Scheller RH, Tsien RW, et al: Ablation of P/Q-type Ca(2+)channel currents, altered synaptic transmission, and progressiveataxia in mice lacking the alpha(1A)-subunit. Proc Natl Acad Sci U S A1999, 96(26):15245–15250.

46. Rajakulendran S, Kaski D, Hanna MG: Neuronal P/Q-type calcium channeldysfunction in inherited disorders of the CNS. Nat Rev Neurol 2012,8(2):86–96.

47. Tottene A, Conti R, Fabbro A, Vecchia D, Shapovalova M, Santello M,van den Maagdenberg AM, Ferrari MD, Pietrobon D: Enhanced excitatorytransmission at cortical synapses as the basis for facilitated spreadingdepression in Ca(v)2.1 knockin migraine mice. Neuron 2009, 61(5):762–773.

48. Tottene A, Fellin T, Pagnutti S, Luvisetto S, Striessnig J, Fletcher C, Pietrobon D:Familial hemiplegic migraine mutations increase Ca(2+) influx throughsingle human CaV2.1 channels and decrease maximal CaV2.1 currentdensity in neurons. Proc Natl Acad Sci U S A 2002, 99(20):13284–13289.

49. Mato S, Lafourcade M, Robbe D, Bakiri Y, Manzoni OJ: Role of the cyclic-AMP/PKA cascade and of P/Q-type Ca++ channels in endocannabinoid-mediatedlong-term depression in the nucleus accumbens. Neuropharmacology 2008,54(1):87–94.

50. Wei F, Li P, Zhuo M: Loss of synaptic depression in mammalian anteriorcingulate cortex after amputation. J Neurosci 1999, 19(21):9346–9354.

51. Harvey J, Palmer MJ, Irving AJ, Clarke VR, Collingridge GL: NMDA receptordependence of mGlu-mediated depression of synaptic transmission in theCA1 region of the rat hippocampus. Br J Pharmacol 1996, 119(6):1239–1247.

52. Vignes M, Clarke VR, Davies CH, Chambers A, Jane DE, Watkins JC,Collingridge GL: Pharmacological evidence for an involvement of group IIand group III mGluRs in the presynaptic regulation of excitatory synapticresponses in the CA1 region of rat hippocampal slices.Neuropharmacology 1995, 34(8):973–982.

53. Kamiya H, Shinozaki H, Yamamoto C: Activation of metabotropic glutamatereceptor type 2/3 suppresses transmission at rat hippocampal mossy fibresynapses. J Physiol 1996, 493(Pt 2):447–455.

54. Burke JP, Hablitz JJ: Presynaptic depression of synaptic transmissionmediated by activation of metabotropic glutamate receptors in ratneocortex. J Neurosci 1994, 14(8):5120–5130.

55. Glaum SR, Slater NT, Rossi DJ, Miller RJ: Role of metabotropic glutamate(ACPD) receptors at the parallel fiber-Purkinje cell synapse. J Neurophysiol1992, 68(4):1453–1462.


56. Calabresi P, Pisani A, Mercuri NB, Bernardi G: Heterogeneity of metabotropicglutamate receptors in the striatum: electrophysiological evidence. Eur JNeurosci 1993, 5(10):1370–1377.

57. Swartz KJ, Bean BP: Inhibition of calcium channels in rat CA3 pyramidalneurons by a metabotropic glutamate receptor. J Neurosci 1992,12(11):4358–4371.

58. Swartz KJ, Merritt A, Bean BP, Lovinger DM: Protein kinase C modulatesglutamate receptor inhibition of Ca2+ channels and synaptic transmission.Nature 1993, 361(6408):165–168.

59. Sahara Y, Westbrook GL: Modulation of calcium currents by a metabotropicglutamate receptor involves fast and slow kinetic components in culturedhippocampal neurons. J Neurosci 1993, 13(7):3041–3050.

60. Sayer RJ, Schwindt PC, Crill WE: Metabotropic glutamate receptor-mediatedsuppression of L-type calcium current in acutely isolated neocorticalneurons. J Neurophysiol 1992, 68(3):833–842.

61. Choi S, Lovinger DM: Metabotropic glutamate receptor modulation ofvoltage-gated Ca2+ channels involves multiple receptor subtypes incortical neurons. J Neurosci 1996, 16(1):36–45.

62. Anwyl R: Metabotropic glutamate receptors: electrophysiologicalproperties and role in plasticity. Brain Res Brain Res Rev 1999, 29(1):83–120.

63. Stefani A, Pisani A, Mercuri NB, Calabresi P: The modulation of calciumcurrents by the activation of mGluRs. Functional implications.Mol Neurobiol 1996, 13(1):81–95.

64. Conn PJ, Pin JP: Pharmacology and functions of metabotropic glutamatereceptors. Annu Rev Pharmacol Toxicol 1997, 37:205–237.

65. Schoepp DD, Jane DE, Monn JA: Pharmacological agents acting atsubtypes of metabotropic glutamate receptors. Neuropharmacology 1999,38(10):1431–1476.

doi:10.1186/1744-8069-9-58Cite this article as: Kang et al.: N-type voltage gated calcium channelsmediate excitatory synaptic transmission in the anterior cingulatecortex of adult mice. Molecular Pain 2013 9:58.

Submit your next manuscript to BioMed Centraland take full advantage of:

• Convenient online submission

• Thorough peer review

• No space constraints or color figure charges

• Immediate publication on acceptance

• Inclusion in PubMed, CAS, Scopus and Google Scholar

• Research which is freely available for redistribution

Submit your manuscript at www.biomedcentral.com/submit

Documents

Kang MOLECULAR PAIN RESEARCH Open Access N-type voltage ... · RESEARCH Open Access N-type voltage gated calcium channels mediate excitatory synaptic transmission in the anterior